Lysyl hydroxylases (LH) catalyze the hydroxylation of specific lysines in collagen that are precursors for the cross-link formation essential for the tensile strength of collagen. The LH family includes a) LH1 in which mutations in the LH1 gene cause enzyme deficiency and result in the inherited connective tissue disorder Ehlers-Danlos syndrome type VI, b) the alternatively-spliced forms of LH2, and c) LH3. Our hypothesis is that the LH isoforms have specificity for hydroxylation of lysines in different collagen domains. The alternative transcripts of LH2(long) and LH2(short) may be specific for the hydroxylation of lysines in the telopeptide (non-helical) and helical collagen domains, respectively. Their relative expression would therefore influence the type and stability of collagen cross-links formed. Pyridinoline cross-links are derived from hydroxylated lysine residues within the telopeptide and are irreversibly increased in fibrotic tissues. The identity of the telopeptide lysyl hydroxylase therefore becomes important to establish. The pattern of lysyl hydroxylation of the telopeptide and helical domains of type I collagen will be analyzed, using mass spectrometry and amino acid analysis, in a recombinant baculovirus system in which viral stocks of type I collagen are expressed together with each LH isoform (LH1, LH2 (long and short) and LH3). Preliminary data shows that the splicing mechanism for the inclusion of exon 13A in the alternatively-spliced long form of LH2 requires a newly-synthesized protein factor. This mechanism is important to establish as recently LH2(long) has been shown to be overexpressed, in parallel with increased pyridinoline cross-links, in fibrotic tissues. We have designed a viable minigene construct that replicates the endogenous pattern of alternate splicing of LH2 observed in HEK293 cells. To identify cis-acting regulatory elements controlling expression of the alternate exon (exon 13A), deletions will be performed in highly conserved regions of the adjacent introns. Potential exon sequence elements that influence the splicing pattern will be defined by mutational analysis. This study should confirm a) that LH2(long) is the telopeptide lysyl hydroxylase that is required for the formation of pyridinoline cross-links that are increased in fibrosis and b) identify the splicing factors controlling the expression of LH2(long), so that potential sites for therapeutic inhibition of LH2(long) that is overexpressed in fibrotic conditions such as keloids and scleroderma may be identified. [unreadable] [unreadable] [unreadable]